A Simple and Flexible Strategy for Single-Cell Proteomic Analysis Based on Protein Immobilization and Digestion Tube Reactor

Mass spectrometry-based single-cell proteomics (SCP) analysis has witnessed rapid development over the past 10 years. However, the current preprocessing methodologies face several challenges: multiple time-consuming steps, reliance on costly consumables and advanced instrumentation, and the necessity for specialized expertise and training, which hinder the widespread application of deep SCP analysis. Here, we develop a simple and flexible strategy that seamlessly integrates single-cell sampling, preprocessing, and liquid chromatography tandem mass spectrometry (LC-MS/MS) injection by constructing a microliter single-cell protein immobilization and digestion tube reactor (SPIDR), which remains free from additional transfer steps. The reactor, made through inner surface functionalization of a commercially available insert tube, achieves the end-to-end single-cell rapid preprocessing within 1 h at a low cost. The microliter reactor with a relatively large volume, instead of the popular nanoliter/picoliter volume, significantly reduces operational difficulty and facilitates process automation. Using the SPIDR workflow, an average of 4186, 3171, and 4018 protein groups are quantified from single A549 cells (n = 16), HeLa cells (n = 16), and MCF-7 cells (n = 16), respectively. Furthermore, we investigate the proteomic heterogeneity of cervical cancer cells at different apoptotic stages following paclitaxel treatment at the single-cell level, demonstrating the potential of single-cell proteomics in addressing biological problems.基于质谱的单细胞蛋白质组学（single-cell proteomics, SCP）分析在过去十年间取得了迅猛发展。然而，当前的预处理方法仍面临诸多挑战：包含多步耗时操作、依赖高成本耗材与高端仪器设备，且需要专业技术知识与操作培训，这些问题制约了深度单细胞蛋白质组学分析的广泛应用。本研究开发了一种简便灵活的策略：通过构建微升级单细胞蛋白质固定与酶解管反应器（single-cell protein immobilization and digestion tube reactor, SPIDR），实现了单细胞采样、预处理与液相色谱串联质谱（liquid chromatography tandem mass spectrometry, LC-MS/MS）进样的无缝整合，且无需额外的样品转移步骤。该反应器通过对市售插入式试管的内表面进行功能化修饰制得，可在1小时内完成端到端的单细胞快速预处理，且成本低廉。相较于当前主流的纳升/皮升体积反应器，该微升级反应器拥有相对更大的体积，可显著降低操作难度，并助力流程自动化。采用SPIDR工作流程，分别对单个人A549细胞（n=16）、HeLa细胞（n=16）与MCF-7细胞（n=16）进行定量分析，平均可鉴定并定量得到4186、3171与4018个蛋白群组。此外，本研究在单细胞层面解析了经紫杉醇处理后处于不同凋亡阶段的宫颈癌细胞的蛋白质组异质性，证实了单细胞蛋白质组学在解决生物学问题方面的应用潜力。